The research proposal is designed to explore the mechanisms of activation of the large molecular weight cytosolic PLA2 (cPLA2). Our hypothesis is that complete activation of cPLA2 requires two phosphorylation/dephosphorylation steps in addition to an increase in [Ca2+]: at serine-505 and at threonine-60. Threonine-60 is contained within the calcium lipid binding domain (CaLB) of cPLA2 and is conserved in all proteins that translocate in response to [Ca2+]. Our Specific Aims are 1.To determine if cPLA2 plays a role in cell injury due to energy depletion by comparing injury in LLC-Pk1 cells which overexpress cPLA2 and by inhibiting cPLA2 expression using oligodeoxynucleotides. 2 To examine the role of phosphorylation in enzyme activation and translocation. 3. To determine the site of phosphorylation of cPLA2. 4. TO evaluate whether p42MAP Kinase associates with cPLA2 intracellularly and whether any other non-kinase proteins interact with cPLA2. These last experiments will be done using a novel two hybrid interaction system. The environment is ideal for the successful completion of this proposal. Dr. Bonventre is very well respected established investigator with areas of expertise in PLA2, cell injury and signal transduction. The methods described are already in use in his laboratory and multiple projects involving PLA2 are in progress in his laboratory.